Liposome compositions and methods of use thereof

ABSTRACT

The present application relates to compositions comprising and methods of using a liposome comprising a pHLIP polypeptide, wherein a lipid bilayer of the liposome is substantially free of the pHLIP polypeptide.

RELATED APPLICATIONS

This application claims the benefit of priority under 35 U.S.C. §119(e)to U.S. Provisional Application NO: 61/373,660, filed Aug. 13, 2010,which is incorporated herein by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention made with U.S. Government support under Grant NumberW81XWH-07-1-0498 from the Department of Defense, and Grant No. R01CA133890 from the National Institutes of Health. The Government hascertain rights in the invention.

INCORPORATION-BY-REFERENCE OF SEQUENCE LISTING

The contents of the text file named “40984-503001US_ST25.txt”, which wascreated on Sep. 16, 2011 and is 20.3 KB in size, are hereby incorporatedby reference in their entirety.

FIELD OF THE INVENTION

The invention relates to compositions and methods for delivery ofmolecules to cells.

BACKGROUND

Despite many advances in the field of cancer diagnosis and treatment, areliable method of identifying and treating cancer cells while sparingnon-cancerous cells has been elusive. One of the limitations is theheterogeneity of human cancers. It has therefore been problematic torely on any single tumor biomarker even for one type of cancer.Selective and efficient targeting and delivery of therapeutic agents totumor cells remains a challenge. As such, there is a pressing need todevelop new strategies for the introduction of various agents to cells.

SUMMARY OF THE INVENTION

The invention is based on the surprising discovery that pH (Low)Insertion Peptide (pHLIP) liposomes target acidic tissue, and releaseliposome content, i.e., cargo into a cell. The compositions and methodsof the invention provide a liposome comprising a pHLIP polypeptide,wherein a hydrophobic region of the lipid bilayer of the liposome issubstantially free of the pHLIP polypeptide. pHLIP is directly attachedto the polar headgroup of the phospholipid or is attached to a polymer(PEG), which in turn is attached to the polar headgroup, but it does notspan the hydrophobic phospholipid tail region of the pHLIP-liposome.Optionally, pHLIP can be attached directly to the lipid bilayer.

In some cases, the pHLIP polypeptide is conjugated to a pharmaceuticallyacceptable polymer (e.g., polymer of polyethylene glycol (PEG),tetrafunctional polyethylene oxide (PEO), polypropylene oxide (PPO), orethylenediamine block copolymer). For example, the pHLIP polypeptide isattached to polymer-phospholipid (e.g., PEG-phospholipid). Thepolymer-phospholipid is attached to a terminal end of the pHLIPpolypeptide. The PEG serves a dual purpose of protecting the liposomeagainst immune destruction (e.g., uptake and clearance by macrophages)and holding the pHLIP away from the lipid bilayer of the liposome.Optionally, the amino-terminal end of the pHLIP is attached to the PEGportion of the PEG-phospholipid, and the carboxy-terminal end of thepHLIP is located outside of liposome's lipid bilayer, i.e., the pHLIPdoes not span the lipid bilayer area of the liposome. Alternatively, areversible peptide such as pHLIP reverse sequence,TEDADVLLALDLLLLPTTFLWDAYRAWYPNQECA (SEQ ID NO: 41), is used. In thatcase, the carboxy-terminal end of the pHLIP is attached to the PEGportion of the PEG-phospholipid, and the amino-terminal end of the pHLIPis located outside the liposome's lipid bilayer. In another example,pHLIP is attached directly to a phospholipid in the lipid bilayer. Ineither example, pHLIP decorates the outside of the liposome.

In some embodiments, the liposome further comprises a cargo inside ofthe liposome or inside of the lipid bilayer. pHLIP liposomes targetdiseased tissue in a pH-dependent manner and release liposome content,i.e., cargo into diseased cells. The cargo may comprise any molecule.For example, the cargo comprises a therapeutic compound, such as a polarcomposition or a non-polar composition. Polar cargo is encapsulatedwithin the liposome, while non-polar cargo is contained in the lipidbilayer of the liposome. Polar cargo molecules include a polar toxin, asmall interfering ribonucleic acid (siRNA), a deoxyribonucleic acid(DNA), a phallotoxin, or a polar inhibitor. Non-polar moleculesincluding non-polar inhibitors are also suitable for the methodsdescribed herein.

The liposome may further comprise a lipid bilayer-tethered cargo. Thetethered cargo is attached to a lipid by a cleavable or non-cleavablebond. In some embodiments, the tethered cargo is attached to a lipid bya S—S bond. In another embodiment, the liposome further compriseshydrophobic cargo incorporated into the lipid bilayer of the liposome.By a “hydrophobic” molecule is meant a molecule having little or noaffinity for water. For example, paclitaxel (Taxol®) is an exemplaryhydrophobic cargo.

Other exemplary cargo molecules include a DNA-binding agent, ceramide,doxorubicin, Doxil® (a pegylated (polyethylene glycol coated)liposome-encapsulated form of doxorubicin), and Myocet™ (a non-pegylatedliposome-encapsulated for of doxorubicin).

Without pHLIP-liposomes, many polar and hydrophobic agents, e.g.,chemotherapeutic/antitumor drugs, are only inefficiently taken up bytarget cells. For example, very polar molecules include sulfonates andphosphonates.

Polar Surface Area (PSA) is a commonly used medicinal chemistry metricfor the optimisation of a drug's ability to permeate cells. Moleculeswith a polar surface area of greater than 140 angstroms squared tend tobe poor at permeating cell membranes. A significant advantage of theinvention is that even compounds that in the past have beencharacterized as being poor at permeating cells are successfullydelivered into cells and released inside the cells usingpHLIP-liposomes.

A method of delivering a cargo into a target cell is carried out bycontacting the target cell with a cargo-loaded pHLIP-decorated (pHLIP⁺)liposome. Preferably, hydrophobic region of the lipid bilayer of thepHLIP⁺ liposome is substantially free of pHLIP polypeptide. The use ofpHLIP-containing liposomes leads to at least 1%, 5%, 10%, 25%, 50%, or2-fold, 5-fold, 10-fold or more of cargo delivered to the cytoplasm ofthe target cell compared to the amount delivered using pHLIP⁻ liposome(i.e., a liposome that does not contain a pHLIP). pHLIP⁺ liposomesdeliver their cargo to cells by fusion with the cell membrane, byendocytosis, or both. The target cell may be characterized by amicroenvironment comprising a low pH.

In some embodiments, the pHLIP⁺ liposome fuses with a cell membrane ofthe target cell. In some cases, the pHLIP⁺ liposome both fuses with acell membrane of the target cell and is taken up by the cell byendocytosis. For example, the pHLIP+ liposome preferentially fuses witha membrane of an endosomal and/or a lysosomal compartment of the targetcell after uptake by endocytosis.

For example, the target cell is a tumor cell or other cell characterizedby a local microenvironment of low pH, e.g., cells of a diseased tissuewith a naturally acidic extracellular environment or cells of a tissuewith an artificially induced acidic extracellular environment relativeto normal physiological pH. Many diseased tissues are characterized byan acidic microenvironment. However, acidity in tumors or non-tumortarget tissues is optionally induced by co-injection of glucose or adiluted solution of acid at the tissue site at which therapy using thecompositions is desired. For example, an acidifying composition (e.g.,glucose or dilute acid) is administered, e.g., injected subcutaneously,before delivery of the pH sensitive compositions (30 s, 1 min., 5 min.,10 min., 30 min., 1 hr., 2 hrs, 6 hrs, 12 hrs, 24 hrs, 48 hrs, or moreprior to administration of the environmentally sensitive composition tothe target tissue site). Alternatively, the tissue acidifying agent andthe pHLIP composition are co-administered. For example, the diseasedtissue is selected from the group consisting of cancer,inflammation/inflamed tissue, ischemia/ischemic tissue, tissue affectedby stroke, arthritis, infection with a microorganism (e.g., a bacteria,virus, or fungus), or atherosclerotic plaques.

pHLIP-liposomes are also useful to deliver therapeutic agents ordiagnostic agents to cell surfaces in a diseased tissue with a naturallyacidic extracellular environment or in a tissue with an artificiallyinduced acidic extracellular environment relative to normalphysiological pH. Administration of a neutralizing agent to an acidicsite, e.g., a bicarbonate solution, is used to reduce pHLIPbinding/insertion and pHLIP labeling or targeting of cells at that site.

Pharmaceutical compositions comprising the liposomal structuresaccording to the present embodiments are also within the invention

pHLIP peptides comprise a membrane sequence that comprises at least 8amino acids. Preferably, the length of the peptide does not exceed 50amino acids (excluding the cargo moiety). pHLIP peptides arecharacterized by pH-dependent membrane-binding or membrane-insertingactivity. A membrane sequence is an amino acid sequence of a peptidethat associates with or inserts into a lipid bilayer. For example, themembrane sequence of the peptide spans a cell membrane structure. Themembrane sequence mediates translocation of a composition (e.g., cargocompounds) that is attached to, e.g., conjugated to, the membranesequence. Translocation means translocation of cargo across a membraneof an artificial lipid bilayer structure and/or that of a cell. Thepeptide component of the composition (e.g., membrane sequence) ismonomeric and non-pore forming, i.e., a peptide comprising the membranesequence does not assemble into a multimeric pore or channel structurein a lipid bilayer or cell membrane. For example, insertion of themembrane sequence of the composition into a lipid membrane does notcause calcium release out of lipid vesicles and does not causehemoglobin leakage out of red blood cells.

The membrane sequence comprises greater than 8 and less than 50residues. Preferably, the range is 13-25 residues. At least 6 of the 8amino acids of the insertion sequence are non-polar. In someembodiments, the 6 non-polar amino acids of the membrane sequence arecontiguous. At least one of the 8 amino acids of the insertion sequenceis protonatable. The protonatable amino acid is located within 10 aminoacids (e.g., within 2, 3, 4, 5, 6, 7, 8, or 9 residues) of the non-polaramino acids (not immediately contiguous to a non-polar amino acid). Thepeptide comprises naturally-occurring amino acids, non-naturallyoccurring amino acids, amino acids that are DNA-encoded as well as thosethat are not encoded by DNA or RNA. The peptide includes L-amino acidsas well as D-amino acids.

All polynucleotides and polypeptides of the invention are purifiedand/or isolated. Specifically, as used herein, an “isolated” or“purified” nucleic acid molecule, polynucleotide, polypeptide, orprotein, is substantially free of other cellular material, or culturemedium when produced by recombinant techniques, or chemical precursorsor other chemicals when chemically synthesized. Purified compounds areat least 60% by weight (dry weight) the compound of interest.Preferably, the preparation is at least 75%, more preferably at least90%, and most preferably at least 99%, by weight the compound ofinterest. For example, a purified compound is one that is at least 90%,91%, 92%, 93%, 94%, 95%, 98%, 99%, or 100% (w/w) of the desired compoundby weight. Purity is measured by any appropriate standard method, forexample, by column chromatography, thin layer chromatography, orhigh-performance liquid chromatography (HPLC) analysis. A purified orisolated polynucleotide (ribonucleic acid (RNA) or deoxyribonucleic acid(DNA)) is free of the genes or sequences that flank it in itsnaturally-occurring state. Purified also defines a degree of sterilitythat is safe for administration to a human subject, e.g., lackinginfectious or toxic agents.

Similarly, by “substantially pure” is meant a nucleotide or polypeptidethat has been separated from the components that naturally accompany it.Typically, the nucleotides and polypeptides are substantially pure whenthey are at least 60%, 70%, 80%, 90%, 95%, or even 99%, by weight, freefrom the proteins and naturally-occurring organic molecules with theyare naturally associated.

An “isolated nucleic acid” is a nucleic acid, the structure of which isnot identical to that of any naturally occurring nucleic acid, or tothat of any fragment of a naturally occurring genomic nucleic acidspanning more than three separate genes. The term covers, for example:(a) a DNA which is part of a naturally occurring genomic DNA molecule,but is not flanked by both of the nucleic acid sequences that flank thatpart of the molecule in the genome of the organism in which it naturallyoccurs; (b) a nucleic acid incorporated into a vector or into thegenomic DNA of a prokaryote or eukaryote in a manner, such that theresulting molecule is not identical to any naturally occurring vector orgenomic DNA; (c) a separate molecule such as a cDNA, a genomic fragment,a fragment produced by polymerase chain reaction (PCR), or a restrictionfragment; and (d) a recombinant nucleotide sequence that is part of ahybridgene, i.e., a gene encoding a fusion protein. Isolated nucleicacid molecules according to the present invention further includemolecules produced synthetically, as well as any nucleic acids that havebeen altered chemically and/or that have modified backbones.

Although the phrase “nucleic acid molecule” primarily refers to thephysical nucleic acid molecule and the phrase “nucleic acid sequence”refers to the sequence of the nucleotides the nucleic acid molecule, thetwo phrases can be used interchangeably.

The transitional term “comprising,” which is synonymous with“including,” “containing,” or “characterized by,” is inclusive oropen-ended and does not exclude additional, unrecited elements or methodsteps. By contrast, the transitional phrase “consisting of” excludes anyelement, step, or ingredient not specified in the claim. Thetransitional phrase “consisting essentially of” limits the scope of aclaim to the specified materials or steps “and those that do notmaterially affect the basic and novel characteristic(s)” of the claimedinvention.

Other features and advantages of the invention will be apparent from thefollowing description of the preferred embodiments thereof, and from theclaims. Unless otherwise defined, all technical and scientific termsused herein have the same meaning as commonly understood by one ofordinary skill in the art to which this invention belongs. Althoughmethods and materials similar or equivalent to those described hereincan be used in the practice or testing of the present invention,suitable methods and materials are described below. All publishedforeign patents and patent applications cited herein are incorporatedherein by reference. Specifically, PCT/US2011/043928, filed Jul. 13,2011 is incorporated herein by reference. Genbank and NCBI submissionsindicated by accession number cited herein are incorporated herein byreference. All other published references, documents, manuscripts andscientific literature cited herein are incorporated herein by reference.In the case of conflict, the present specification, includingdefinitions, will control. In addition, the materials, methods, andexamples are illustrative only and not intended to be limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic presentation of pHLIP-mediated fusion orendocytotic uptake of liposomes. The triangle represents the carboxy(COOH) end of the pHLIP, which is most distant from the lipid bilayer ofthe liposome. The amino (NH₂) end of pHLIP is attached to the PEGportion of the PEG-phospholipid.

FIG. 2 is a series of line graphs, a dot plot, a bar chart, andschematics showing that pHLIP-induced inter-liposome fusion in low pHsolution. pHLIP-mediated inter-liposome fusion was studied by Octadecylrhodamine B (R18) self-quenching assay. Liposomes labeled with R18 weremixed with various concentrations of unlabeled liposomes (POPC). Duringthe process of dropping pH of solution, rhodamine fluorescence wasmonitored on the spectrofluorometer. The rhodamine fluorescence ofpHLIP-liposomes (DOPE-pHLIP or DOPC-pHLIP) increase significantly afterdropping pH (FIGS. 2A1 and 2A2). 200 uM of R18-labeled liposome wasmixed with 6 mM of unlabeled POPC. 200 uM of R18-labeled liposome wasmixed with elevated concentration of unlabeled POPC (0-6 mM; FIG. 2B).The amount of PEG-lipid or pHLIP-conjugated lipid containing in liposomeaffects the percentage of fusion (FIG. 2C). Schematic of the lipidfusion assay (FIG. 2D). Schematic of the liposome composition (FIG. 2E).

FIG. 3 is a series of bar charts demonstrating that pHLIP promotescellular uptake of liposomes. Cellular uptake of pHLIP-decoratedliposomes containing different fluorescent lipids (Rho-FA(R18),Rhodamine-PE, Fluorescein-DHPE) in comparison with the uptake of thesame liposomes without pHLIP is shown.

FIG. 4 is a series of bar charts showing cellular uptake ofpHLIP-decorated liposomes at neutral and low pHs. pHLIP promotescellular uptake of liposomes at low pH.

FIG. 5 is a bar chart demonstrating that cellular uptake of liposomeincreases with increase of amount pHLIP on the surface of liposome (DOPClipids were used in study).

FIG. 6 is a bar chart showing cellular uptake of liposomes decoratedwith different amounts of PEG polymer (DOPC lipids were used in study).

FIG. 7 is a bar chart showing the results of a trypan-blue fusion assay.After treatment of liposomes containing Fluorescein-lipid, thefluorescein fluorescence of cells was counted before and after addingtrypan blue. Cell-impermeable trypan blue can quench fluoresceinfluorescence only if fluorescein dye is located on the outer leaflet ofcellular membrane facing to the extracellular space, which might occuronly in a result of liposome-cell membrane fusion.

FIG. 8 is a series of bar charts showing the results of an endocytosisassay. Cell-liposome incubation was done for 15 or 60 min at 4° C. or37° C. in different media (PBS, DMEM, ATP-depletion medium). Lowtemperature and ATP-depletion medium were used to reduce endocytoticuptake.

FIG. 9 is a series of photomicrographs demonstrating cellularlocalization of fluorescent fatty acids (R18) incorporated intoliposomes containing PEG polymers and pHLIP or no pHLIP on the surface(non-fusogenic DOPC lipids were used in study). In case of liposomes,fluorescent signal was mostly localized in endosomes, while pHLIPpromotes distortion of plasma and endosome membranes and release ofR18-labeled FA into cytoplasm and targeting of mitochondria. All imagesare taken from live cells under inverted fluorescence microscopy at 60×objective magnification.

FIG. 10 is a series of photomicrographs showing cellular localization offluorescent lipids (Rho-PE) incorporated into liposomes containing PEGpolymers and pHLIP or no pHLIP on the surface (non-fusogenic DOPC lipidswere used in study). In case of liposomes, fluorescent signal was mostlylocalized in endosomes, while pHLIP promotes distortion of plasma andendosome membranes and release of lipids into cytoplasm. All images aretaken from live cells under inverted fluorescence microscopy at 60×objective magnification.

FIG. 11 is a series of photomicrographs showing cellular localization offluorescent lipids (FITC (fluorescein)-PE) incorporated into liposomescontaining PEG polymers and pHLIP. FIGS. 11A and 11B are phase contrastand fluorescent images of cells treated with FITC-liposome-pHLIP.Staining of plasma membrane is evident. Co-localization of fluoresceinfluorescence (FIG. 11C) and plasma membrane staining of red-fluorescentAlexa Fluor594 wheat germ agglutinin (FIG. 11D).

FIG. 12 is a series of photomicrographs showing pHLIP mediated releaseof PI from liposomes. The propidium iodide (PI) was encapsulated inFluorescein-labeled liposomes. 10 nmol of liposome were incubated withcells attached to the collagen in 100 uL of low pH media for 1 hr at 37C.

FIG. 13 is a series of photomicrographs showing that pHLIP promotesliposome uptake in low pH extracellular environment of tumors. Liposomescontaining Rho-PE lipids, were given as a single intro-tumoral injectioninto mice with tumors established by subcutaneous injection of HeLa-GFPcancer cells. Mice were sacrificed at 24 hours post-injection, andtumors were collected. Whole-body and tumor images were taken on KodakImager.

FIG. 14 is a schematic diagram showing that pHLIP(pH-Low-Insertion-Peptide) insertion into membrane occurs as a result ofprotonation of Asp/Glu residues due to a decrease of pH. Protonationenhances peptide hydrophobicity and increases its affinity for a lipidbilayer, which triggers peptide insertion and formation of transmembranehelix. Since many pathological states are associated with thedevelopment of elevated level of extracellular acidity (or lowextracellular pH) pHLIP could be used for selective delivery ofdiagnostic and therapeutic agents to the cancer cells. Attachment ofcargo molecules to the N-terminus.

FIG. 15 is a schematic showing that liposomes are artificial vesiclesprimarily composed of phospholipid bilayers. Liposomes can be filledwith drugs, and used to deliver drugs for cancer and other diseases.pHLIP technology may be used for selective delivery of liposomes tocancer cells.

FIG. 16 shows two types of liposomes (100 nm in diameter), one of whichwas carrying 5 mol % of pHLIP peptides and the other was not. Bothliposomes contained 5 mol % of nanogold-lipids and 10 mol % PEGylatedlipids. Cells were treated with two types of liposomes separately,washed, fixed. After fixation, cells were treated with silverenhancement solution and analyzed under the light microscope.Nanogold-lipids were mostly localized on the plasma and nuclearmembranes of cells treated with pHLIP-liposomes

FIG. 17A is a series of photomicrographs showing pHLIP mediated cellstaining by nanogold. HeLa-GFP cells were incubated with pHLIP-nanogoldand nanogold particles at neutral and low pHs, washed, fixed andenhanced by silver then visualized under light microscope. The highestuptake was observed at low pH in presence of pHLIP.

FIG. 17B is a series of photomicrographs demonstrating nanogoldparticles distribution in tumors. Tumor sections collected from micethat received a single iv injection of pHLIP-nanogold. Nanogoldparticles were treated with silver enhancement solution and visualizedunder the microscope. Nanogold particles delivered to tumor by pHLIPwere localized on cancer cells identified by GFP fluorescence.

FIG. 18 provides an illustration of a liposome of one embodiment inwhich the lipid bilayer of the liposome is comprised of a polymer-pHLIP(e.g., PEG-pHLIP) is anchored into the liposomal membrane. The polymeris attached to the N-terminus of the pHLIP polypeptide. The pHLIPpolypeptide is wholly outside the lipid bilayer.

FIG. 19A is a photomicrograph of a Cryo-TEM image of DOPE liposomes.FIG. 19B is a photomicrograph of a Cryo-TEM image of DOPE-pHLIPliposomes. FIG. 19C is a photomicrograph of a Cryo-TEM image of DOPCliposomes. FIG. 19D is a photomicrograph of a Cryo-TEM image ofDOPC-pHLIP liposomes.

FIG. 20 is a series of photomicrographs showing pHLIP liposome cellularuptake. The light images (FIGS. 20A and 20C) and fluorescent images(FIGS. 20B and 20D) were taken after 4 days incubation.

FIG. 21 is a series of photomicrographs showing ER labeling,mitochondria staining, and R18-liposome uptake in A549 cell suspensiontreated with R18 containing liposome.

FIG. 22 is a series of photomicrographs of cryo-TEM images ofceramide-containing liposomes.

FIG. 23A is a line graph showing the diameter of control-liposome andpHLIP-liposome are 104 nm and 125 nm, respectively. FIG. 23B is a dotplot showing that the size of control-liposome is stable after 3 days,while the size of pHLIP-liposome increases slightly.

FIG. 24A-B are scans showing that there is no fusion of liposomes ifpHLIP is not attached to the surface (no increase of fluorescence at lowpH). FIG. 24B shows that there is increase of fluorescence whenpHLIP-coated liposomes are mixed with POPC liposomes, which indicatesfusion of liposomes. Thus, pHLIP promotes fusion only at low pH. FIG.24C is a line graph showing a summary of the data in FIGS. 24A and 24B.The black line shows no increase of fluorescence for the controlexperiment, and the red line shows that fluorescence increases in caseof pHLIP-coated liposomes.

FIG. 25 is a schematic of pHLIP-mediated delivery of liposomal ceramideto cells.

FIG. 26 is a schematic of pHLIP-mediated delivery of liposomal ceramideto a cell suspension.

FIGS. 27A and 27B are a series of bar charts demonstrating the resultsof pHLIP-mediated delivery of liposomal ceramide to cell suspension.Delivery of ceramide (C6) using pHLIP-liposomes lead to a significantlygreater amount of cell death at low pH compared to the level of celldeath at high pH. Moreover, ceramide (C6) pHLIP-liposomes lead to asignificantly greater amount of cell death at low pH compared toceramide liposomes alone (in the absence of pHLIP) at low pH.

FIG. 28 is a diagram of states I, II, and III of pHLIP.

FIG. 29 is a schematic depicting the hydrophilic polar headgroup regionand hydrophobic phospholipid tail region of a liposome.

FIG. 30 is a line graph showing verification of reaction product bySELDI-TOF mass spectrometry.

DETAILED DESCRIPTION OF THE INVENTION

The invention is based on the discovery that pHLIP-liposomes target theacidic microenvironment of a tissue, and release liposome content, i.e.,cargo, into a cell. Because pHLIP does not insert into cellularmembranes at normal pH, pHLIP allows for the selective delivery of cargomolecules to diseased tissue with low extracellular pH by preventing theentry of cargo molecules into a healthy cell.

Prior to the invention described herein, the release of liposome contentinto cells was problematic due to the entrapment of liposomes and theircontents within the endosomal compartments after endocytosis. Asdescribed herein, pHLIP promotes the fusion of liposomes with cellularmembranes or the fusion of liposomes with endosome membranes afterendocytotic uptake of pHLIP liposomes, thereby releasing the contents ofthe liposomes into the cell. These two mechanisms of action areillustrated in FIG. 1.

The hydrophobic region of the lipid bilayer of an exemplarypHLIP-liposome is substantially free of the pHLIP polypeptide. pHLIP isdirectly attached to the polar headgroup of the phospholipid or isattached to a polymer (PEG), which in turn is attached to the polarheadgroup, but pHLIP peptide does not span the hydrophobic phospholipidtail region of the pHLIP-liposome. Alternatively or optionally, pHLIP isattached directly to the lipid bilayer. A schematic depicting thehydrophilic polar headgroup region and hydrophobic phospholipid tailregion of a liposome is provided in FIG. 29.

In some cases, pHLIP liposomes deliver molecules to the inside of a cellby inserting into a cellular lipid bilayer and transporting C-terminalcargo molecules across the plasma membrane. Any molecule is a suitablecargo molecule. Exemplary functional cargo molecules include peptidenucleic acid (PNA), phalloidin, doxorubicin, and paclitaxel.

Peptide nucleic acid (PNA) is an artificially synthesized polymersimilar to DNA or RNA; however, the backbone of PNA is composed ofrepeating N-(2-aminoethyl)-glycine units linked by peptide bonds. Sincethe backbone of PNA contains no charged phosphate groups, the bindingbetween PNA and DNA strands is stronger than between DNA/DNA strands dueto the lack of electrostatic repulsion. In this manner, PNA acts as agene regulation agent by exhibiting antisense activity. Although PNAitself has poor membrane permeability, pHLIP liposomes significantlyenhance its translocation and antisense activity.

Phalloidin, a cytotoxin isolated from the Death Cap mushroom Amanitaphalloides, is a polar, cell-impermeable, cyclic heptapeptide (An etal., 2010 PNAS, 107(47): 20246-20250). Because phalloidin iscell-impermeable, prior to the invention described herein, phalloidinwas not suitable for therapeutic purposes. As described herein, pHLIPliposomes deliver phalloidin into the cytoplasm of cells, therebypreventing cell migration and metastasis.

Doxorubicin intercalates DNA, and is commonly used in the treatment of awide range of cancers. Similarly, paclitaxel (Taxol®) is a mitoticinhibitor used in cancer chemotherapy. pHLIP liposomes selectivelydeliver cancer agents into the cytoplasm of diseased cells with lowextracellular pH. In this manner, drug efficacy is enhanced, and theside effects of anti-cancer therapy are reduced.

Numerous pHLIP peptide sequences are described in WO 2006/078816 A2,herein incorporated by reference. The invention is based on thesurprising discovery that a liposome comprising a pHLIP peptide isuseful for enhanced delivery of agents (particularly agents that aredifficult to deliver using other methods) to target cells characterizedby a low pH microenvironment, e.g., tumor cells.

As described above, an acidic environment triggers insertion of pHIPinto synthetic lipid bilayer structures or cellular membrane in vitroand in vivo. As described herein, since acidity is associated with manypathological states, including cancer, pHLIP is used as adisease-targeting acid-specific peptide. Described herein is theselective delivery of gold nanoparticles and pHLIP liposomes to cancercells in vivo. Gold nanospheres and nanorods are used for theenhancement of radiation therapy and for thermal ablation of tumors.

A major challenge is to selectively deliver enough gold material tocancer cells to produce the desirable effect. The compositions andmethods of the invention overcome the drawbacks and challengesassociated with previous methods. The in vivo data described hereinshows high uptake of pHLIP-labeled liposomes by cancer cells andefficient delivery of cargo to such cells.

Liposomal Structures

The liposomes of some embodiments comprise polymer-phospholipids (e.g.,PEG-phospholipid). In some embodiments, the pHLIP polypeptide isattached to polymer-phospholipid (e.g., PEG-phospholipid). Thepolymer-phospholipid may be attached at the terminal end of the pHLIPpolypeptide. In some embodiments, the amino-terminal end of pHLIP isattached to the PEG-phospholipid and the carboxy-terminal end of pHLIPis located outside of the liposome.

In some embodiments, the bilayer of the liposome comprises at least 1,2, 5, 8 or 10% polymer (e.g., at least 15%, at least 20%, at least 25%,at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, atleast 80%, at least 90%, at least 95%). In some embodiments, the innerlipid bilayer contains less than 40% of total polymer (less than 30%,25%, 20%, 15%, 10%, or 5%). In some embodiments, the outer lipid bilayercontains at least 60% of total polymer (more than 60%, 65%, 70%, 75%,80%, 90%, or 95%) contained in the liposome.

In some embodiments, the bilayer of the liposome comprises at least 10%PEG (e.g., at least 15%, at least 20%, at least 25%, at least 30%, atleast 40%, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, at least 95%). In some embodiments, the inner lipid bilayercontains less than 40% of total PEG (less than 30%, 25%, 20%, 15%, 10%,or 5%). In some embodiments, the outer lipid bilayer contains at least60% of total PEG (more than 60%, 65%, 70%, 75%, 80%, 90%, or 95%)contained in the liposome. The PEG may have a molecular weight of MWabout 350, 550, 750, 1000, 2000, 5000, or 10000.

pHLIP promotes the following activities: endocytotic uptake of liposomeswith up to 10 mol % of PEG polymer (5 mol % is on the surface ofliposome in the other leaflet) at low pH; disruption of endosome andlysozome and release of lipids from liposome and liposomal content intocytoplasm; fusion between cell membranes and lipid bilayer of liposomeat low pH; delivery of R18 (Rhodamine-fatty acid) to mitochondria in lowpH extracellular environment; release of DNA-targeting dye PI (propidiumiodide) encapsulated into liposome; delivery of gold nanoparticles tointernal cellular membranes at low pH. R18 has affinity to mitochondriamembrane, but when delivered by regular liposomes it could not reachmitochondria. Using pHLIP-liposomes, such compounds, and in fact, anymitochondria-targeting compound are readily delivered to intracellularorganelles such as mitochondria. In another example, pH-dependentdelivery of any polar compounds, e.g., polar agents used for imaging ortherapy suitable for encapsulation in liposomes, and any non-polarmolecules optionally trapped within lipids of liposome is enhanced usingpHLIP-liposomes compared to conventional liposomes (i.e., liposomes thatdo not contain pHLIP).

pHLIP Sequences

Tables 1-2 provide a summary of exemplary pHLIP sequences used inpHLIP-liposomes. Table 1 includes long pHLIP sequences. The sequences ofTable 1, if they insert into a membrane, go across with their C-terminusand leave N-terminus in the extracellular space.

TABLE 1 Name Sequence WT-1a GEQNPIYWARYADWLFTTPLLLLDLALLVDADEGSEQ ID NO: 1 WT-1b ACEQNPIYWARYWARYADWLFTTPLLLLDLALLVDADEGT SEQ ID NO: 2WT-1 GGEQNPIYWARYADWLFTTPLLLLDLALLVDADEGT SEQ ID NO: 3 WT-2ACEQNPIYWARYADWLFTTPLLLLDLALLVDADET SEQ ID NO: 4 WT-Cys1AAEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTCG SEQ ID NO: 5 WT-Cys2AEQNPIYWARYADWLFTTPLLLLDLALLVDADEGCT SEQ ID NO: 52 WT-Cys3GGEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTCG SEQ ID NO: 6 Cys-WT1ACEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTG SEQ ID NO: 7 Cys-WT2ACEQNPIYWARYADWLFTTPLLLLDLALLVDADEGT SEQ ID NO: 8 Lys-WTAKEQNPIYWARYADWLFTTPLLLLDLALLVDADEGT SEQ ID NO: 9 WT-KCAAEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTKCG SEQ ID NO: 10 K-WT-CAKEQNPIYWARYADWLFTTPLLLLDLALLVDADECT SEQ ID NO: 11 N-pHLIPACEQNPIYWARYANWLFTTPLLLLNLALLVDADEGTG SEQ ID NO: 12 K-pHLIPACEQNPIYWARYAKWLFTTPLLLLKLALLVDADEGTG SEQ ID NO: 13 NNQGGEQNPIYWARYADWLFTTPLLLLDLALLVNANQGT SEQ ID NO: 14 D25AAAEQNPIYWARYADWLFTTPLLLLALALLVDADEGT SEQ ID NO: 15 D14AAAEQNPIYWARYAAWLFTTPLLLLDLALLVDADEGT SEQ ID NO: 16 P20AAAEQNPIYWARYADWLFTTALLLLDLALLVDADEGT SEQ ID NO: 17 D25EAAEQNPIYWARYADWLFTTPLLLLELALLVDADEGT SEQ ID NO: 18 D14EAAEQNPIYWARYAEWLFTTPLLLLDLALLVDADEGT SEQ ID NO: 19 3DAAEQNPIIYWARYADWLFTDLPLLLLDLLALLVDADEGT SEQ ID NO: 20 R11QGEQNPIYWAQYADWLFTTPLLLLDLALLVDADEGTCG SEQ ID NO: 21 D25UpGGEQNPIYWARYADWLFTTPLLLDLLALLVDADEGTCG SEQ ID NO: 22 D25DownGGEQNPIYWARYADWLFTTPLLLLLDALLVDADEGTCG SEQ ID NO: 23 D14UpGGEQNPIYWARYDAWLFTTPLLLLDLALLVDADEGTCG SEQ ID NO: 24 D14DownGGEQNPIYWARYAWDLFTTPLLLLDLALLVDADEGTCG SEQ ID NO: 25 P20GAAEQNPIYWARYADWLFTTGLLLLDLALLVDADEGT SEQ ID NO: 26 H1DDDEDNPIYWARYADWLFTTPLLLLHGALLVDADE C T SEQ ID NO: 27 H2DDDEDNPIYWARYAHWLFTTPLLLLHGALLVDADEG C T SEQ ID NO: 28 H2NDDDEDNPIYWARYAHWLFTTPLLLLHGALLVNADE C T SEQ ID NO: 29 H2N2DDDEDNPIYWARYAHWLFTTPLLLLHGALLVNANE C T SEQ ID NO: 30 1a-TrpAEQNPIYWARYADFLFTTPLLLLDLALLVDADET SEQ ID NO: 31 1b-TrpAEQNPIYFARYADWLFTTPLLLLDLALLVDADEGT SEQ ID NO: 32 1c-TrpAEQNPIYFARYADFLFTTPLLLLDLALLWDADET SEQ ID NO: 33 Fast-1 AKEDQNPYWARYADWLFTTPLLLLDLALLVDG SEQ ID NO: 34 Cys-Fast1 ACEDQNPYWARYADWLFTTPLLLLDLALLVDG SEQ ID NO: 35 Fast1-CysAEDQNPYWARYADWLFTTPLLLLDLALLVDCG SEQ ID NO: 36 Fast1-E-CysAEDQNPYWARYADWLFTTPLLLLELALLVE CG SEQ ID NO: 37 Fast2 AKEDQNPYWRAYADLFTPLTLLDLLALWDG SEQ ID NO: 38 Cys-Fast2 ACEDQNPYWRAYADLFTPLTLLDLLALWDG SEQ ID NO: 39 Fastest AKEDQNDPYWARYADWLFTTPLLLLDLALLVG SEQ ID NO: 40

Table 2 includes sequences termed short and medium pHLIP sequences. Theyall insert in membrane in a pH-dependent manner, while they do not haveC-terminal flanking sequence. Double underline indicates residues (Cysor Lys), which are used to conjugate pHLIPs with cargo molecules. pHLIPsequences contain L-amino acids; alternatively, the pHLIP comprisesD-amino acids.

TABLE 2 Name Sequence WT-reverse TEDADVLLALDLLLLPTTFLWDAY SEQ ID NO: 41RAWYPNQECA Sh AEQNPIYW ARYADWLFTTPL SEQ ID NO: 42 Sh-CysAEQNPIYW ARYADWLFTTP C L SEQ ID NO: 43 Cys-Sh A C EQNPIYW ARYADWLFTTPLSEQ ID NO: 44 Sh-1Trp AEQNPIYFARYADWLFTTPL SEQ ID NO: 45 Sh-1DKEDQNPWARYADLLFPTTLAW SEQ ID NO: 46 Cys-Sh-1D ACEDQNPWARYADLLFPTTLAWSEQ ID NO: 47 Cys-Med-2D ACEDQNPWARYADWLFPTTLLLLD SEQ ID NO: 48Cys-Sh-1E ACEEQNPWARYAELLFPTTLAW SEQ ID NO: 49 Cys-Med-2EACEEQNPWARYAEWLFPTTLLLLE SEQ ID NO: 50 Cys-Med-3EACEEQNPWARYLEWLFPTETLLLEL SEQ ID NO: 51

DNA-encoded and non-coded amino acids are described below in Table 3.Additional non-natural amino acids that can be used are known in theart, e.g., as described in Hendrickson et al., 2004, Ann. Rev. Biochem.73:147-176; hereby incorporated by reference.

TABLE 3 Coded and Non-Coded Amino Acids NO: abbrev name^(s) 1 Alaalanine 2 Arg arglnine 3 Asn asparagine 4 Asp aspartic acid 5 Cyscysteine 6 Gin glutamine 7 Glu glutamic acid 8 Gly glycine 9 Hishistidine 10 Ile isoleucine 11 Leu leucine 12 Lys lysine 13 Metmethionine 14 Phe Phenylalanine 15 Pro proline 16 Ser serine 17 Thrthreonine 18 Trp tryptophan 19 Tyr tyrosine 20 Val valine 21 AcpaAminocaprylic acid 22 Aecys (S)-2-aminoethyl-L-cysteine•HCI 23 AfaAminophenyl acetate 24 Aiba -aminoisobytyric acid 25 Aile alloisoleucine26 AIg L-allylglycine 27 Aba amlnobutyric acid 28 Aphep-aminophenylalanine 29 Bat -alanine 30 Brphe p-bromophenylalanine 31Cha cyclohexylalanine 32 Cit citrulline 33 Clala -chloroalanine 34 Ciecycioleucine 35 Clphe p-chiorophenylalanine 36 Cya cysteic acid 37 Dab2,4-diamino-butyric acid 38 Dap 2,3-diaminopropionic acid 39 Dhp3,4-dehydro-proline 40 Dhphe 3,4-,dihydroxy-phenyl-alanine 41 Fphep-fluorophenylalanine 42 Gaa D-glucose-aminic acid 43 Hag Homo-arginine44 Hlys hydroxyl-lysine•HCI 45 Hnvl DL-hydroxynorvaline 46 HogHomoglutamine 47 Hoph homophenylalanlne 48 Has homoserine 49 Hprhydroxyl-proline 50 Iphe p-lodophenylalanine 51 Ise isoserine 52 Mle-methyl-leucine 53 Msmet DL-methionine-s-methylsulfo-niumchloride 541Nala 3-(1-naphthyl)alanine 55 2Nala 3-(2-naphthyl)alanine 56 Nlenorleucine (or 2-aminohexanoic acid) 57 Nmala N-methyl-alanine 58 Nvanorvaline (or 2-aminopentanoic acid) 59 Obser 0-benzylserine 60 Obtyr0-benzyl-tyrosine 61 Oetyr O-ethyltyrosine 62 Omser O-methylserine 63Omthr 0-methyt-hreonine 64 Omtyr 0-methyl-tyrosine 65 Orn ornithine 66Pen penicillamlne 67 Pga pyroglutamic acid 68 Pip pipecolic acid 69 Sarsarcosine 70 Tfa 3,3,3-trifluoroalanine 71 Thphe 6-hydroxydopa 72 VigL-vinylglycine 73 Aaspa(−)-(2R)-2-amino-3-(2-aminoethylsulfonyl)propanoic acid dihydrochloride74 Ahdna (2S)-2-amino-9-hydroxy-4,7-dioxanonanolc acid 75 Ahoha(2S)-2-amino-6-hydroxy-4-oxahexanoic acid 76 Ahsopa(−)-(2R)-2-amino-3-(2-hydroxyethylsulfonyl)propanoic acid

A “conservative amino acid substitution” is one in which the amino acidresidue is replaced with an amino acid residue having a similar sidechain. Families of amino acid residues having similar side chains havebeen defined in the art. These families include amino acids with basicside chains (e.g., lysine, arginine, histidine), acidic side chains(e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g.,glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine),nonpolar side chains (e.g., alanine, valine, leucine, isoleucine,proline, phenylalanine, methionine, tryptophan), beta-branched sidechains (e.g., threonine, valine, isoleucine) and aromatic side chains(e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a residuein a pHLIP sequence (corresponding to a location relative to SEQ ID NO:3) is replaced with another amino acid residue from the same side chainfamily.

pHLIP Peptide is Monomeric

pHLIP peptides, e.g., (SEQ ID NO: 4) are a water-soluble polypeptidesbased on the bacteriorhodopisin C helix, which was found to insertacross a membrane to form a stable transmembrane alpha helix. Peptidefolding and membrane insertion are driven by a drop of pH from neutralor high (>7.4) to slightly acidic (7.0-6.5 and less) pHs. The apparentpK of insertion was found to be 6.0. pHLIP is a monomer in each of itsthree major states: unstructured and soluble in water (state I) atneutral pH, unstructured and bound to the surface of a membrane atneutral pH (state II), and inserted across the membrane as an α-helix atlow pH (state III). In contrast, all pore forming peptides first formaggregates on the membrane surface and then “fall” into membrane andform pores. Thus, an additional advantage of theenvironmentally-sensitive compositions is their monomeric nature, e.g.,they do not require assembly into a multimeric suprastructure like poreformers.

Delivery of Cargo Using pHLIP-Liposomes

Although gold nanospheres and nanorods have been used for theenhancement of radiation therapy and for thermal ablation of tumors,delivery of enough gold material to cancer cells to produce thedesirable effect has been a challenge. Gold nanoparticles attached tothe N-terminus of pHLIP have been successfully delivered to tumors andaccumulate on the surface of membrane of cancer cells. Another even moreefficient way to deliver gold material (or other compounds) to tumor (orother cells characterized by low pH) is to use pHLIP-liposomes.pHLIP-liposomes are useful deliver to cells in a pH-dependent manner anycompound, e.g., polar or hydrophobic compounds, that have been difficultto get into cells using other methods. In contrast to fusogenicliposomes developed before for delivery, which can fuse with cellularmembrane only in the absence of PEG coating, pHLIP can mediate fusionbetween lipid bilayer of plasma membrane or membrane ofendosome/lysozome and liposomes made of non-fusogenic lipids andcontaining 10 mol % of PEG. pHLIP conjugated to the pegylated liposomespromotes pH-modulated: i) endocytotic uptake of liposomes by targetedcell, distortion of endosome compartment and release of lipids orliposome content into cytoplasm; and ii) direct liposomal fusion withplasma membrane and release of liposomal content into cytoplasm. pHLIPpromotes mitochondrial delivery of R18 incorporated into liposome. Asdescribed in detail below, various assays were performed on liposomes insolution and on live cells to demonstrate that pHLIP mediates uptake ofliposomes. The in vivo data shows high uptake of pHLIP-labeled liposomesby cancer cells.

In some embodiments, the present invention relates to the use of pHLIPtechnology for selective delivery of gold nanoparticles and liposomes tocancer cells in vivo. The data described herein demonstrate that goldnanoparticles attached to the N-terminus of pHLIP are delivered totumors and accumulate on the surface of membrane of cancer cells.Distribution of gold nanoparticles in tumor was investigated by lightmicroscopy after silver enhancement.

Toxicity

Toxicity is one of the most critical issues in the selection of anydelivery agent. For example, the use of pore-forming membrane peptidesas delivery agents is complicated by the toxicity associated with theformation of pores in cellular membranes in vivo. By contrast, theinteraction of pHLIP with liposomes and cellular membranes at bothneutral and low pHs does not lead to membrane leakage, and no cellulartoxicity was seen over a range of peptide concentrations.

Selectivity of Targeting

The pH-dependent interaction of pHLIP with membranes allows selectivityin the targeting of acidic (less than pH 7.0) diseased tissue. As notedabove, acidity and hypoxia are considered as universal cancerbiomarkers, and pHLIP is used as an acidity-targeting probe. Besidescancer, many other pathological states, such as inflammation, ischemia,stroke, arthritis and others are characterized by acidity in theextracellular space, which may broaden the potential applications ofpHLIP. In vivo fluorescence imaging in mice and rats demonstrated thatpHLIP can target acidic tissues, such as kidneys, tumors of varioussizes and origins, and anatomical sites of inflammation, e.g.,arthritis, infection, atherosclerotic plaques. In addition tofluorescence imaging, PET (positron emission tomography) imaging of theacidic environment in human prostate tumors was performed using⁶⁴Cu-DOTA conjugated to pHLIP. PET studies demonstrated that theconstruct avidly accumulated in LNCaP and PC-3 tumors and that tumoruptake correlates with the differences in the bulk extracellular pH(pH_(e)) measured by MR spectroscopy. To manipulate the acidity oftissues, a buffer solution is administered to the subject systemicallyor local to the area in which a pH change is desired. In this manner,pHLIP-liposome-mediated delivery of a cargo compound is regulated, e.g.,reduced or stopped. For example, administering bicarbonated water, whichincreases tissue pH, results in a reduction of tumor targeting by pHLIP.

Molecular Mechanism of pH-Dependent Membrane Insertion of pHLIP

The transmembrane (TM) part of exemplary pHLIP peptides contain two Aspresidues. At neutral pH these charged residues enhance peptidesolubility and serve as anchors keeping the peptide at the surface ofmembrane, thereby preventing pHLIP partitioning into the hydrophobicmembrane bilayer. A reduction of pH induces protonation of Asp residues,and as a result, the overall hydrophobicity of the peptide increases,enhancing the affinity of the peptide for the lipid bilayer core andtriggering peptide folding and insertion. The replacement of the key Aspresidues by Lys, Ala or Asn leads to the loss of peptide of pH-dependentmembrane insertion, as measured in liposomes, red blood cells andconfirmed by in vivo fluorescence imaging. The K-pHLIP peptide, wherethe two Asp residues in the transmembrane region are replaced with Lysresidues, does not demonstrate tumor targeting. The Ala substitutionsyield a peptide that aggregates in solution (but de-aggregates when itbecomes diluted in bodily fluids or tissue upon administration to asubject), while the Lys and Asn substitutions give peptides that are toopolar to insert either at neutral or low pH. The replacement of one ofthe Asp residues in the TM part of the peptide by a Glu residue resultsin a shift of pH of membrane insertion from 6.0 to 6.5. Replacement ofboth Asp residues by Glu results in enhancement of peptide aggregationand formation of elements of secondary structure on the bilayer surfaceat neutral pH (see Tables 1 and 2).

Data obtained using liposomes, cultured cells and mice confirmed thatthe mechanism of membrane entry of pHLIP is not mediated by endocytosis,interactions with cell receptors or pore formation; rather, themechanism is the formation of a helix across the lipid bilayer,triggered by the increase of peptide hydrophobicity due to theprotonation of negatively charged residues induced by low pH.

Solubility and Stability of pHLIP in Blood

Poor solubility due to aggregation is a typical property of membranepeptides, which has complicated studies and applications. Isolated orpurified pHLIP, as any membrane peptide, also has a tendency toaggregate, especially at high concentrations and/or low pH. However, inaqueous solution at neutral pH pHLIP exists as a monomer atconcentrations less than 30 μg/mL (˜7.0 μM), as studied by fluorescenceand CD spectroscopy measurements, size exclusion chromatography coupledwith “on-line” laser light scattering, ultraviolet and refractive indexdetection (SEC-LS/UV/RI) and analytical ultracentrifugation experiments.When the solubility of the peptide is compromised as a result ofmutations, the affinity of the peptide for a membrane and its overallconformational properties change. Thus, studies were undertaken todesign pHLIP peptides that are optimized for clinical diagnostic andtherapeutic use.

The oligomeric state of the peptide on the surface of a membrane (stateII) and inserted into the lipid bilayer (state III) were evaluated byFRET performed with two different donor-acceptor probes attached to theN-terminus of the peptide. The data demonstrate that, at lowconcentrations, the peptide is monomeric in both states II and III (FIG.28).

Peptide interactions with proteins, especially plasma proteins, andmembranes determine the pharmacokinetics of the peptide at neutral pH.pHLIP demonstrates prolonged circulation in the blood (several hours),which is consistent with its ability to bind weakly to membrane surfacesat neutral and high pH, preventing the rapid clearance by the kidneyexpected for a small, soluble peptide. pHLIP binding to membranes isdriven by hydrophobic interactions. If the peptide sequence were mademore hydrophobic, tighter binding to red blood cells and epithelialcells and more aggregation in solution, and slower clearance and reducedbioavailability would occur. Making the peptide less hydrophobicaccelerates clearance and prevents the peptide from finding its targets.Therefore, fine tuning of the solubility is an important property tooptimize pHLIP performance in vivo.

Another important property is the stability of peptides in the blood,since proteases in the serum can degrade peptides consisting of L-aminoacids within minutes. While polypeptides made from D-amino acids aremuch more stable, they are often unsuitable for specific receptorbinding applications as a consequence of their altered chirality. Sincethe mechanism of pHLIP involves relatively nonspecific interactions witha fluid lipid bilayer, pHLIP peptides composed of L- or D-amino acidsdemonstrate the same biophysical and tumor targeting properties. Thisobservation confirms the evidence that the pHLIP targeting does notrequire any specific molecular binding event. The only conspicuousdifference is that D-pHLIPs form left-handed helices across membranesrather than the right-handed helices formed by L-pHLIPs.

EXAMPLES Example 1 pHLIP-Mediated Delivery of Liposomes

The following regents and methods were used to generate the datadescribed herein.

Lipids DOPE 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine

DOPC 1,2-dioleoyl-sn-glycero-3-phosphocholine

DSPE-PEG(2000) Maleimide1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethyleneglycol)-2000] (ammonium salt)

DSPE-PEG(2000)1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000]

Fluorescein DHPEN-(fluorescein-5-thiocarbamoyl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine,triethylammonium salt

Rhod PE 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissaminerhodamine B sulfonyl

R18 (Octadecyl Rhodamine B Chloride)

Conjugation of pHLIP with Lipids

pHLIP containing Cys on the N-terminus and DSPE-PEG(2000) Maleimidelipids. Standard maleimide chemistry reaction was applied in methanol.(Reaction of thiol with maleimide.)

whereR¹—DSPE-PEG(2000) Maleimide (MW 2941)R²—cys-pHLIP (MW 4150)R¹:R²=1:1.5 in DMF or 1:1.2 in Methanol.

The reaction product was verified by SELDI-TOF masspec. Expected mass ofabout 7 kDa was observed (FIG. 30).

Liposome Compositions Used in Experiments with Cells

DOPC: DOPC 85 mol % DSPE-PEG 5-10 mol % Fluorescent-lipids 5 mol %DOPC-pHLIP: DOPC 85 mol % DSPE-PEG 0-5 mol % Fluorescent-lipid 5 mol %DSPE-PEG-pHLIP 0-5 mol % DOPE: DOPE 85 mol % DSPE-PEG 5-10 mol %Fluorescent-lipid 5 mol % DOPE-pHLIP: DOPE 85 mol % DSPE-PEG 0-5 mol %Fluorescent-lipid 5 mol % DSPE-PEG-pHLIP 0-5 mol %Liposome Preparation

Liposomes were prepared by the thin film method (extrusion). Achloroform solution of the desired lipids (1 μmol) was evaporated usingrotary evaporator, producing an even, thin film. The film was placedunder a vacuum overnight to remove trace solvent impurities. This filmwas then hydrated in 1 mL 10 mM phosphate, 150 mM NaCl stock buffersolution via 10 freeze-thaw-vortex cycles. The resulting multilamellarliposome solution was then extruded 15 times through 100 nmpolycarbonate filters and sterilized by filtering through 0.2 μm filter.

Cryo-Electron Microscopy

Cryo-electron microscopy (cryo-EM) is a form of transmission electronmicroscopy (TEM) where the sample is studied at cryogenic temperatures(generally liquid nitrogen temperatures). It allows the observation ofspecimens that have not been stained or fixed in any way, showing themin their native environment.

FEI Vitrobot™ Mark IV is a fully automated vitrification robot forplunge-freezing of aqueous (colloidal) samples. For sample preparation,vitrification in liquid ethane was performed via Vitribot apparatus,with a single blot of 3 sec, an offset of −1, and drain and wait time of1 sec. For imaging, sample was kept at −175° C. during imaging in a JEOL2100 TEM with an accelerating voltage of 200 kV. Images were taken at20,000× and 40,000×.

Cryo-TEM images of liposomes (DOPE, DOPE-pHLIP, DOPC, and DOPC-pHLIP)are shown in FIG. 19.

Inter-Liposome Fusion Assay

pHLIP-mediated inter-liposome fusion was studied by an Octadecylrhodamine B (R18) self-quenching assay. Liposomes labeled with R18 weremixed with various concentrations of unlabeled liposomes (POPC). Duringthe process of dropping pH of solution, rhodamine fluorescence wasmonitored on the spectrofluorometer (FIG. 2).

The spectrofluorometer instrument was utilized. Slow kinetics—emissionintensity measurement (excitation/emission: 556 nm/590 nm).

Steps:

The rhodamine fluorescence of pHLIP-liposomes (DOPE-pHLIP or DOPC-pHLIP)increased significantly after dropping pH from 8 to 4. 200 μM ofR18-labeled liposome was mixed with 6 mM of unlabeled POPC (FIG.2A1-2A2). 200 μM of R18-labeled liposome was mixed with elevatedconcentration of unlabeled POPC (0-6 mM) at pH 4 (FIG. 2B). The amountof PEG-conjugated lipid containing in liposome affects the percentage offusion (FIG. 2C). For FIG. 2C, 200 μM of R18-labeled liposome was mixedwith 6 mM of unlabeled POPC. The percentage calculations are as follows:Percentage of

${{fusion} = {\frac{{FL}_{pH} - {FL}_{0}}{{FL}_{MAX} - {FL}_{0}} \times 100\%}},$where FL₀ is initial fluorescent intensity of liposome mixture at pH 8,FL_(pH) is fluorescent intensity of liposome mixture at pH 4, FL_(MAX)is fluorescent intensity of liposome mixture at pH 4 after freeze-thawcycle.

The scheme of the fusion assay and liposome composition used in studyare provided in FIGS. 2D and 2E.

Cell Suspension

Trypsinized cells were counted using a hemacytometer and diluted to2×10⁵ cells/ml in serum-free low pH media. 20 nmol liposomes wereincubated with 1×10⁵ cells in 500 μL serum-free low pH media for 15 minor 1 hr at 4° C. or 37° C. The cells were then pelleted bycentrifugation (2000 rpm, 4 min) at 4° C. or 37° C. The cells wereresuspended in 500 μL fresh serum-free low pH media and centrifuged asecond time. This second pellet was resuspended in 100 μL same media.The cells were counted using cellometer: The sample was mixed well, and2 μL of trypan blue was added to 18 μL of sample. 20 μL, of thissolution was loaded into disposable counting chamber (slide). Thechamber was inserted into cellometer, and software was used to countcells. The cells were reseeded in collagen-coated cell dish formicroscopy imaging.

Example 2 pHLIP Enhances Uptake of Liposomes by Cells

pHLIP (pH-Low-Insertion-Peptide) insertion into membrane occurs as aresult of protonation of Asp/Glu residues due to a decrease of pH.Protonation enhances peptide hydrophobicity and increases its affinityfor a lipid bilayer, which triggers peptide insertion and formation oftransmembrane helix. Since many pathological states are associated withthe development of elevated level of extracellular acidity (or lowextracellular pH), pHLIP-liposomes are ideally suited for selectivedelivery of diagnostic and therapeutic agents to the cancer cells.Attachment of cargo molecules to the N-terminus (FIG. 14).

One approach to deliver gold material (or cytotoxic compounds) to tumoris to use liposomes. In contrast to fusogenic liposomes developed beforefor delivery, which can fuse with cellular membrane only in the absenceof PEG coating, pHLIP mediates fusion between lipid bilayer of plasmamembrane or membrane of endosome/lysozome and liposomes made ofnon-fusogenic lipids and containing 10 mol % of PEG. pHLIP conjugated tothe pegylated liposomes promotes pH-modulated: i) endocytotic uptake ofliposomes by targeted cell, distortion of endosome compartment andrelease of lipids or liposome content into cytoplasm; and ii) directliposomal fusion with plasma membrane and release of liposomal contentinto cytoplasm. pHLIP promotes mitochondrial delivery of R18,incorporated into liposome. pHLIP was found to mediate uptake ofliposomes. The in vivo data demonstrate high uptake of pHLIP-labeledliposomes by cancer cells.

Cell uptake studies were performed as follows: cells in suspension weretreated with 40 uM of fluorescently labeled pHLIP-liposomes (DOPE-pHLIPor DOPC-pHLIP) and control-liposomes (DOPE or DOPC) under differentconditions, after washing, fluorescence of cells was counted bycellometer. (a) Liposomes containing different fluorescent lipid(Rho-FA(R18), Rhodamine-PE, Fluorescein-DHPE) were tested (FIG. 3).pHLIP-liposomes show high cell uptake. (b) Cells were incubated withR18-labeled liposomes in different pH of medium (FIG. 4). Liposomecontaining different amount of pHLIP-conjugated lipids (c; FIG. 5) andPEG-lipids (d; FIG. 6) were also investigated. (e) Trypan blue quenchingassay: After treatment of liposomes containing Fluorescein-lipid, theFITC-fluorescence of cells was counted before and after adding trypanblue (FIG. 7). Cell-impermeable trypan blue can quench FITC fluorescenceonly if FITC dye is located on the outer leaflet of cellular membranefacing to the extracellular space, which might occur only in a result ofliposome-cell membrane fusion. (f) Endocytosis assay: cells insuspension were incubated with liposomes for 15 or 60 min at 4° C. or37° C. in different media (PBS, DMEM, ATP-depletion medium). Lowtemperature and ATP-depletion medium are used to reduce endocytoticuptake. The results are presented in FIG. 8. Thus, the data indicatedthat pHLIP enhanced uptake of liposomes by cells, and the primarypathway of liposome uptake was endocytosis.

A549 cell suspension (10×10⁵) was treated with R18 containing liposome(20 nmol) in 500 μL of serum-free low pH media for 1 hour at 37 C. Thecells were pelleted by centrifugation (2000 rpm, 4 min) and resuspendedin fresh DMEM. The cells were reseeded in collagen-coated cell dishes(FIG. 20). The light images (a, c) and fluorescent images (b, d) weretaken after 4 days incubation. pHLIP-containing liposome (d) showed muchhigher cell uptake than the control liposome (b), which did not containpHLIP.

Example 3 pHLIP Promotes Distortion of Plasma and Endosome Membranes,the Release of R18-Labeled FA into the Cytoplasm, and Targeting ofMitochondria

After cellometer counting, cells were reseeded in collagen-coated celldishes for microscopy imaging. Cellular localization of fluorescentfatty acids (R18) incorporated into liposomes containing PEG polymersand pHLIP or no pHLIP on the surface (non-fusogenic DOPC lipids wereused in study). In case of liposomes, fluorescent signal was mostlylocalized in endosomes, while pHLIP promotes distortion of plasma andendosome membranes and release of R18-labeled FA into cytoplasm andtargeting of mitochondria. FIG. 9 shows the localization of Rho-labeledliposome in cells.

FIG. 11 shows images of Fluorescein-labeled liposomes fused with acellular membrane. FIG. 11( a) phase contrast; (b) FITC. Co-localizationof FITC-liposome (c) and plasma membrane staining of red-fluorescentAlexa Fluor594 wheat germ agglutinin (d). The data demonstrate thatlipids are exchanged as a result of fusion with the plasma membrane ormembrane of the endosomal compartment, thereby reaching the plasmamembrane. Thus, the methods described herein promote the delivery andrelease of agents that are encapsulated inside a pHLIP-liposome orattached to lipids of the pHLIP-liposome to the cytoplasm of a cell.

FIG. 12 shows the results of a liposome encapsulation experiment(delivery of propidium iodide to the nucleus). The propidium iodide (PI;4 mM) was encapsulated in Fluorescein-labeled liposomes (FIG. 12). 10nmol of liposome were incubated with cells attached to thecollagen-coated cell dish in 100 μL of low pH media for 1 hr at 37° C.The release of PI from pHLIP-liposomes was observed.

FIG. 16 shows two types of liposomes (100 nm in diameter), one of whichwas carrying 5 mol % of pHLIP peptides and the other was not. Bothliposomes contained 5 mol % of nanogold-lipids and 10 mol % PEGylatedlipids. Cells were treated with two types of liposomes separately,washed, fixed. After fixation, cells were treated with silverenhancement solution and analyzed under the light microscope.Nanogold-lipids were mostly localized on the plasma and nuclearmembranes of cells treated with pHLIP-liposomes (FIG. 16).

A549 cell suspension (10×10⁵) was treated with R18 containing liposome(20 nmol) in 500 uL of PBS (pH6.2) for 15 min at 37 C. The cells werepelleted by centrifugation (2000 rpm, 4 min) and resuspended in freshDMEM. Then the cells were reseeded in collagen-coated cell dishes. After4 days incubation, endoplasmic reticulum (ER) and mitochondria werelabeled by fluorescent dyes of ER-Tracker and Mito-Tracker,respectively. The fluorescent images were taken with the filter settingof GFP, Cy5 and TRITC, corresponding to ER labeling, mitochondriastaining and R18-liposome uptake (FIG. 21).

Example 4 pHLIP Promotes Uptake of Liposome in Low pH ExtracellularEnvironment of Tumors

Liposomes, containing Rho-PE lipids, were given as a singleintra-tumoral injection into mice with tumors established bysubcutaneous injection of HeLa-GFP cancer cells. Mice were sacrificed at24 hours post-injection, and tumors were collected. Whole-body and tumorimages were taken on Kodak in vivo imaging system. As shown in FIG. 13,pHLIP promoted liposome uptake in low pH extracellular environment oftumors, following IV injection of the fluorescent- and gold-containingliposomes.

HeLa-GFP cells were incubated with pHLIP-nanogold and nanogold particlesat neutral and low pHs, washed, fixed and enhanced by silver thenvisualized under light microscope. The highest uptake was observed atlow pH in presence of pHLIP (FIG. 17A). Tumor sections collected frommice received single iv injection of pHLIP-nanogold and nanogoldparticles were treated with silver enhancement solution and visualizedunder the microscope. Nanogold particles delivered to tumor by pHLIPwere localized on cancer cells identified by GFP fluorescence (FIG.17B).

These data indicate that pHLIP-liposomes demonstrate enhanced uptake bycells in environments characterized by low pH (pH<7) compared toliposomes that do not contain pHLIP.

Example 5 pHLIP-Mediated Delivery of Liposomal Ceramide to Cancer Cells

An exemplary ceramide formulation is provided below. Cryo-TEM images ofceramide-containing liposomes are shown in FIG. 22. The size and shape(round) of the particles indicate that ceramide liposomes were formed.

Control-liposome pHLIP-liposome DOPC  37 mol %  37 mol % DOPE 17.5 mol%  17.5 mol %  DSPE-PEG₂₀₀₀ 7.5 mol % 2.5 mol % DSPE-PEG₂₀₀₀-pHLIP   0mol %   5 mol % C8-PEG₇₅₀ 7.5 mol % 7.5 mol % C6-ceramide  30 mol %  30mol % R18 0.5 mol % 0.5 mol %Liposome Size Measurement

The size of liposome was measured by using Dynamic Light Scattering(Zetasizer Nano ZS). The diameters of control-liposome andpHLIP-liposome are 104 nm and 125 nm, respectively (FIG. 23A). After 3days monitoring, the size of control-liposome is stable, while the sizeof pHLIP-liposome increased slightly (FIG. 23B).

Inter-Liposome Fusion Assay of Ceramide Liposome

The fusion assay was performed as described in Example 1 and FIG. 2D.

The results are presented in FIG. 24. FIG. 24A shows that there is nofusion of liposomes if pHLIP is not attached to the surface (no increaseof fluorescence at low pH). FIG. 24B shows that there is an increase offluorescence when pHLIP-coated liposomes are mixed with POPC liposomes,which indicates fusion of liposomes. Thus, pHLIP promotes fusion only atlow pH. FIG. 24C is a summary of FIGS. 24A and 24B. The black line showsno increase of fluorescence for control experiment, and the red lineshows that fluorescence increases in case of pHLIP-coated liposomes. Themethods for this experiment were described in relation to FIG. 2 above;however, the amount of Rho-FA is much less (0.5 mol %) for FIG. 24 thanFIG. 2 (5 mol %). Therefore, the increase of fluorescence is much lessin FIG. 24 comparison to FIG. 2.

A schematic of pHLIP-mediated Delivery of Liposomal Ceramide to Cells isprovided in FIG. 25.

A schematic of pHLIP-mediated delivery of liposomal ceramide to cellsuspension is provided in FIG. 26. FIGS. 27A and 27B are a series of barcharts demonstrating the results of pHLIP-mediated delivery of liposomalceramide to cell suspension. Delivery of ceramide (C6) usingpHLIP-liposomes led to a significantly greater amount of cell death atlow pH compared to the level of cell death at high pH. Moreover,ceramide (C6) pHLIP-liposomes led to a significantly greater amount ofcell death at low pH compared to ceramide liposomes alone (in theabsence of pHLIP) at low pH.

OTHER EMBODIMENTS

While the invention has been described in conjunction with the detaileddescription thereof, the foregoing description is intended to illustrateand not limit the scope of the invention, which is defined by the scopeof the appended claims. Other aspects, advantages, and modifications arewithin the scope of the following claims.

The patent and scientific literature referred to herein establishes theknowledge that is available to those with skill in the art. All UnitedStates patents and published or unpublished United States patentapplications cited herein are incorporated by reference. All publishedforeign patents and patent applications cited herein are herebyincorporated by reference. Genbank and NCBI submissions indicated byaccession number cited herein are hereby incorporated by reference. Allother published references, documents, manuscripts and scientificliterature cited herein are hereby incorporated by reference.

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims.

What is claimed is:
 1. A liposome comprising a pHLIP polypeptide,wherein a hydrophobic region of the lipid bilayer of said liposome issubstantially free of said pHLIP polypeptide, wherein said lipid bilayercomprises a polyethylene glycol (PEG)-phospholipid, and wherein saidpHLIP polypeptide is attached to said PEG-phospholipid.
 2. The liposomeof claim 1, wherein the amino-terminal end of said pHLIP polypeptide isattached to said PEG-phospholipid and the carboxy-terminal end of saidpHLIP polypeptide is located outside of said liposome.
 3. The liposomeof claim 1, wherein the carboxy-terminal end of said pHLIP polypeptideis attached to said PEG-phospholipid, and the amino-terminal end of saidpHLIP polypeptide is located outside of said liposome.
 4. The liposomeof claim 1, wherein said liposome further comprises a cargo inside ofsaid liposome or inside of said lipid bilayer.
 5. The liposome of claim4, wherein said cargo comprises a therapeutic compound.
 6. The liposomeof claim 4, wherein said cargo comprises a polar composition.
 7. Theliposome of claim 1, wherein said liposome further comprises ahydrophobic cargo incorporated into said lipid bilayer.
 8. The liposomeof claim 1, wherein said liposome further comprises a lipidbilayer-tethered cargo.
 9. The liposome of claim 8, wherein saidtethered cargo is attached to a lipid by a cleavable or non-cleavablebond.
 10. The liposome of claim 8, wherein said tethered cargo isattached to a lipid by a S—S bond.
 11. A method of delivering a cargointo a target cell comprising contacting said target cell withcargo-loaded pHLIP+ liposome according to claim 1, a lipid bilayer ofsaid liposome being substantially free of said pHLIP polypeptide,wherein at least 10% more of said cargo is delivered to the cytoplasm ofsaid target cell compared to the amount delivered using pHLIP-liposome.12. The method of claim 11, wherein said target cell is characterized bya microenvironment comprising a low pH.
 13. The method of claim 11,wherein said pHLIP+ liposome fuses with a cell membrane of said targetcell.
 14. The method of claim 11, wherein said pHLIP+ liposome bothfuses with a cell membrane of said target cell and is taken up by saidcell by endocytosis.
 15. The method of claim 11, wherein said pHLIP+liposome preferentially fuses with a membrane of an endosomalcompartment and a lysosomal compartment of said target cell after uptakeby endocytosis.
 16. The method of claim 11, wherein said target cell isa tumor cell, ischemic cell, inflamed cell, bacterially-infected cell,fungus-infected cells, or virally-infected cell.
 17. The method of claim11, wherein said cargo comprises ceramide, a deoxyribonucleotide (DNA)binding agent, a small interfering ribonucleic acid (RNA), a DNA, apolar toxin, an inhibitor, TAXOL®, or doxorubicin.
 18. A pharmaceuticalcomposition comprising the liposome of claim 1.